In situ dextrose production in crude amylaceous materials



United States Patent 3,249,512 .IN SITU DEXTROSE PRODUCTION IN CRUDEAMYLACEQUS MATERIALS Harold E. Bode, 14170 Onaway, Cleveland, Ohio NoDrawing. Filed May 28, 1963, Ser. No. 283,7d5 7 Claims. (Cl. 19531) Thisapplication is a continuation-in-p-art of my copending applicationSerial No. 38,716, filed June 27, 1960, now abandoned.

This invention relates to an improved method of producing dextrose insitu amylaceous edible food or feed products.

Most food products are used in conjunction with sugars such as dextrose,sucrose, invert sugars and the like.

The starch in amylaceous foods is capable of being hydrolyzed by eitherenzyme or acid catalysts into sugars such as dextrose or maltose. Since,in most cases, the cost of the starch in an amylaceous food isconsiderably less than the prevailing costs for a refined sugar such assucrose or dextrose, food technologists have long searched for practicalmeans of converting some or all of the starch in a food material todextrose without deleteriously affecting the protein and fat materialswhich are always present in amylaceous foods such as cereal flours, manyvegetables and some fruits.

In the past, attempts to convert some of the starch in a food to a sugarcomposition that could qualitatively compete with a sugar liquorobtained by dissolving crystalline, refined dextrose or sucrose inwater, have always ended in failures because of the inability to producea sugar product in situ in the cereal or vegetable Withoutsimultaneously producing sufiicient hydrolysis of the protein and fatpresent in the amylaceous food product to cause extremely undesirabletaste or flavor effects.

If amylaceous food products such as wheat flour, corn flour, peas, beansor bananas are subjected to acid startch hydrolyzing agents, the starchis converted to mixtures of dextrose, maltose, dextrins and some bittertasting polysaccharides, such as gentiobiose. Simultaneously, the sameacid catalyst acts on the protein material in the food product toproduce bad tasting protein break-down products such as solublepolypeptides and free amino acids.

These protein hydrolytic break-down products are not only objectionablebecause of their own ill-tasting effects, but are also responsible forthe subsequent production of sugar-addition products which also possesobjectionable taste and flavor properties. These sugar-addition productsresult from the interaction of reducing sugars such as dextrose ormaltose with amino acids or polypeptides to produce nitrogenous sugarderivatives that contribute toward severe color formation and furtherdeleterious taste and flavor effects.

In addition to the bad effect of the protein present in the food, andacid catalyst also acts on the fat to cause some kind of fat hydrolysiswhereby deleteriously tasting fatty material is liberated.

In view of the above difficulties, it is not possible to consider anacid catalyst as a practical means for the in situ production of sugarin an amylaceous food material.

The same principles and objections apply when ordinary diastatic enzymesare used for the purpose of hydrolyzing the starch in the food to sugarmaterial. Ordinary diastases contain protease and lipase enzymeimpurities which causes, during an amylase startch conversion, somehydrolysis of the protein and fat present in the amylaceous foodproduct.

The protease present in ordinary commercial malt or fungal amylases canbe objectionable from both a flavor or a food product physical structurestandpoint. Thus, in the case of the processing of wheat flour intobread, the physical structure of the wheat gluten is important 3,249,512Patented May 3, 1966 "ice to the structure of the finished baked bread;and the hydrolysis of this wheat gluten by any protease would result inpoor quality finished bread products, particularly from a physicalstructure standpoint.

In addition to the presence of proteases and lipases, most commercialdiastases are inefiicient producers of sweetening sugars. Ordinary maltdisease converts or hydrolyzes the starch in the food product to amixture of maltose and dextrins. Maltose has relatively littlesweetening powers and the diastatically produced dextrins haveconsiderably less sweetening effects than maltose. The only enzymeproducible sugar which can be considered to be of practical value in anin situ process for obtaining sugar from an amylaceous food product isthe sugar known as dextrose. This sugar is about three-fourths as sweetas sucrose. However, in addition to the sweetness factor, sugars arefrequently used in food products for other purposes, particularly as afood or substrate for fermenting microorganisms which ferment sugars tosuch materials as ethanol, acetic acid or lactic acid.

From a sugar fermentability standpoint, dextrose is one of the mostefficient sugars, and is superior to maltose or sucrose. Because ofthis, dextrose is the preferred sugar in food processing involving astep including the fermentation of a sugar composition. Examples of foodindustries wherein the use of dextrose is highly desirable from afermentation standpoint, are the baking industries, particularly breadbaking and the brewing industry.

Since as above explained, dextrose is an ideal sugar for use in foodmanufacture, the ideal objective in any kind of in situ process forcreating sugar during the manufacture of a food product is to have acondition wherein a starch hydrolyzing catalyst produces from starchexclusively dextrose, without any kind of simultaneous hydrolysis ofprotein or fat material which may be present in the food product.

Special diastases capable of producing dextrose from certainpolysaccharide materials are known to the art. However, these dextroseproducing enzymes are unsatisfactory for this invention because of thepresence of enzymes impurities such as proteases, lipases, and enzymescapable of causing the polymerization of liberated dextrose into sugarpolymers which have a bitter taste and which cause a loss of dextroseyield.

In accordance with this invention, the preferred dextrose producingenzyme to be used in an in situ sugarproducing process is an enzymesubstantially devoid of any protease, lipase, or transglucosidaseactivity. Such an enzyme is an amylase known as amyloglucosidase. Thisenzyme has the unique property of being capable of breaking down thestarch polysaccharide units exclusively into free dextrose.

Amyloglucosidase is, therefore, an ideal enzyme for in situ dextroseproduction during the processing of an amylaceous food product. However,prior art enzyme products containing amyl-oglucosidase have been unableto fulfill the conditions required for a satisfactory in situ producingfood product process because of the presence of objectionable otherenxyme fractions, such as proteases, lipases and transglucosidase.

The absence of significant amounts of transglucosidase becomes ofparticular importance when an amylaceous material is subjected to aprolonged amyloglucosidase action, such as is the case when the creationof large amounts of in stiu dextrose are desired. Under such prolongedenzyme converting conditions, the dextrose liberated byarnyloglucosidase would tend to be polymerized by the transglucosidaseimpuritie into poor tasting sugar polymers. An ordinary crude fungalenzyme liquor, such as an amylase liquor prepared from Aspergillusniger, contains, in addition to amyloglucosidase, substantial amounts oftransglucosidase, protease and some lipase. Such an enzyme producthasbeen used for the treatment of acidhydrolyzed refined starch liquors forthe purpose of increasing the dextrose content in a product such as cornsyrup. In such a process, the said crude enzyme mixture can operatesatisfactorily because of the absence of pro tein and fat impurities inthe acid converted refined starch liquor. For the treatment of anamylaceous food product containing protein and fat, the use of such afungal enzyme would be impractical One object of this invention is toproduce an enzyme converted amylaceous food product, wherein the soleend product from an amylase conversion is dextrose.

Another object of this invention is to provide a process wherein anamylase-treated amylaceous food product has some of its starch convertedin situ solely to dextrose and wherein none of the non-starch materialsin the said food are modified by the said amylase.

Still another object of the invention is to modify amylaceous materialintended for subsequent carbohydrate fe rmentation, by an enzymatic insitu formation of dextrose from the starch within said amylaceousmaterial, without simultaneously enzymatically modifying the protein,fat, or the insitu liberated dextrose of said treated amylaceousmaterial. Y

A further object of the invention is to provide -a means for theenzymatic in situ production of dextrose from the starch in anamylaceous food without substantially affecting the taste or flavorcontributed by the non-starch materials in the said food product.

A still further object of the invention is to enzymatical- 1y produce insitu dextrose in an amylaceous food under conditions wherein the saidresulting dextrose will have the, same taste or fermenting effects asthat obtained by the separate addition of crystalline dextrose to thesaid food product.

Another object of the invention is to provide an improved process forproducing bread or other bakery products, wherein dextrose is producedfrom the dough by the in situ action of amyloglucosidase added to thedough,

said enzyme containing neither sufficient protease to weaken materiallythe dough structure, nor transglucosidase to produce bitter,non-fermentable dextrose polymers, nor enzyme impurities that adverselyaffect the odor and taste of the baked goods.

Other objects of the invention will, in part, be obvious and will, inpart, appear hereinafter.

For the purposes of this invention, the preferred dextrose producingamylase is a refined fungal amyloglucosidase. It is known thatamyloglucosidase is present in certain varieties of amylases. An amylasesource which is known to contain substantial amounts of amyloglucosidaseis fungal extract obtained from certain fungi, such as, for example,Aspergillus niger or Aspergillus phoenicis.

For the purposes of my invention, I prefer to use a refinedamyloglucosidase obtained by isolation from a crude Aspergillus nigerextract of high amyloglucosidase content. Such crude amyloglucosidaseextracts are known to the prior art as materials which can serve as aprac-,

tical reagent for the conversion of refined starch to dextrose solutionsand the subsequent crystallization of pure dextrose from the saiddextrose liquors.

This crude amyloglucosidase reagent is, however, unsuitable for thepurposes of my invention because of the presence of proteases, lipases,transglucosidase and musty odor ingredients originating from the fungi.In order to be able to practice my invention, it is necesary to subjectthis crude amyloglucosidase fungal extract liquor to a re finingtreatment which isolates the amyloglucosidase and removes substantiallyall protease, lipase, and transglucosidase impurities.

The refining of crude amyloglucosidase can be carried out by methodsknown to the prior art. Thus, selective inorganic adsorbents such assilicates or bentonite, selective inorganic salt solutions such assodium chloride or ammonium sulfate, and selective liquid organicsolvents may be used in various combinations to attain the necessarydegree of amyloglucosidase purity.

In the case of organicsolvents, any selective organic liquid solventwhich dissolves amyloglucosidase, but does not dissolvetransglucosidase, protease, lipase or other amylases; and simultaneouslyis incapable of irreversibly inactivating amyloglucosidase, would besuitable selective solvents.

Examples of selective organic solvents are acetone and isopropanol.Their use for amyloglucosidase refining is discussed by Underkofler andVicente, Iowa State College Journal of Science, volume 30, page 445(1956). The use of bentonite for amyloglucosidase refining is also discussed by these authors. Dirks and Miller describe the separation ofproteases from a-mylases by adsorbing the proteases on aluminumsilicate, Cereal Chemistry, March 1949, pages 98109. US. Patent No.2,121,459 describes the removal of proteases from amylase in enzymemixtures obtained from malted grains, molds, or bacteria. This isaccomplished by the selective adsorption of protease with variousaluminum oxides, such as bauxite.

In general, any reagent or reagent combination which does notdeleteriously affect. the enzymatic activity or the Water solubility ofamyloglucosidase, but which acts as a selective material to separatenon-amyloglucosidase enzymes from non-refined amyloglucosidase, may beused to produce a refined amyloglucosidase which is capable offunctioning in accordance with the principles set forth by thisinvention. The particular combination of refining reagents and refiningsteps which would be used to obtain an amyloglucosidase of satisfactorypurity will depend upon the individual economy of the production of thecrude amyloglucosidase liquor.

A description will now be submitted of the quality requirements whichare necessary for the successful application of amyloglucosidase in thepractice of this invention. Certain testing procedures will be given soas to provide means for better evaluating some ,of the significantfeatures of this invention.

Amyloglucosidase units.These are determined by hydrolyzing starch with aknown quantity of the enzyme under conditions optimum foramyloglucosidase activity. The results are expressed as grams ofdextrose (D) in the hydrolysate, minus the reducing value of theoriginal starch sol (calculated as dextrose), (S), divided by threetimes the number of grams or cc. (E) of the enzyme prep-aration used;

In order to provide a convenient laboratory method to determineamyloglucosidase units of activity, the time of hydrolysis arbitrarilyis limited to a set value of 180 minutes. Accordingly, the amount ofenzyme added in the test is limited to an amount considerably less thanthat amount which would hydrolyze the starch sample completely in thistime.

For the test hydrolysis, five grams of Lintners soluble starch areheated in about. 50cc. of water until solution is complete. Acetatebuffer is added to adjust the solution to pH 4.0. The solution is thenmadeup to a volume of 100 cc. at 60 C. 'a'nd'a small carefully measuredquantity of the enzyme is added. After exactly minutes at 60 C. analiquot is removed and dextrose is determined, as for example, by thewell known Schoorl method, calculating the reducing value as dextrose. Asimilar determination is run on another identically prepared starchsolution, except that no enzyme is added.

As above indicated, given a longer hydrolysis time, the enzyme additioncan usually hydrolyze more starch to dextrose than is indicated by theAmyloglucosidase Units test. Thus, for example, an amyloglucosidasesample containing one unit of activity will ordinarily hydrolyzeactually about 5 to 10 grams of starch almost completely to dextrose ifgiven suflicient time, e about 48 to 72 hours, and particularly if moreconcentrated solutions of starchy substance are used for the hydrolysis.

Transglucosidase activity.-This is determined by measuring the amount ofnon-fermentable products synthesized under conditions favorable fortransglucosidase and amyloglucosidase activities, using maltose as theinitial substrate.

Twenty grams of pure maltose are dissolved in 39 cc. of water to whichare then added 5 cc. of anormal sodium acetate-acetic acid buffer at pH4.0. An amount of enzyme preparation is added which contains 2 units ofamyloglucosidase activity and the mixture is allowed to react for 72hours at 60 C. The reaction flask is then placed in a boiling water bathfor minutes, cooled, adjusted to pH 4.8 with normal sodium hydroxide anddiluted to about 200' cc. Ten grams of dry yeast are added and themixture is fermented for 3 hours at 30 C. The fermentation mixture isthen made up to a volume of 250 cc. and then centrifuged to remove theyeast. .Then 50 cc. aliquots are heated with 5 cc. of about 9 N HCl in aboiling water bath for 3 hours in order to hydrolyze the non-fermentablediand polysaccharides to dextrose. The hydrolyzate is neutralized withNaOH and made up to 100 cc. in a volumetric flask. Dextrose isdetermined, as by the Schoorl method.

A control is also run, adding none of the enzyme preparation under testand no maltose, but using grams of dextrose instead of the maltose.

The dextrose value found after acid hydrolyzing the yeast fermentationliquor, minus the dextrose value found in the control, gives the amountof non-fermentables synthesized by transglucosidase that is in thealiquot. From this the total amount of non-fermentables can becalculated. This value, divided by 2, is the transglucosidase activityin the enzyme preparation, in grams of non-fermentables synthesized perunit of Amyloglucosidase.

Dextrose equivalent (D.E.).This is total reducing sugares calculated asdextrose. It is determined here by a modified Fehling "est, such asdescribed by W. J. Fetzer, Analytical Chemistry, vol. 24, pp. 11291137(1952).

True dextr0se.-By this is meant the actual percentage, dry basis, ofdextrose. A suitable method is described by Sichert and Blyer, Z. Anal,Chem., vol. 107, pp. 328 et seq. (1936).

As will be pointed out more specifically with reference to the workingexamples the amylaceous materials which are operable as startingmate-rials for the instant invention may be selected from a broad classof either purified or crude starchy products. For example, refined cornor wheat starch may be used, as well as potato starch, sweet potatostarch, or like materials. Crude starches from the corn, wheat or potatosource may also be used. Various streams from both wet and dry cornmilling processes may be used as a starting material for the instantinvention. These streams include such materials as starch liquors,hominy feed, hominy grits, ground whole corn, corn flour, brewers gritsor wet cereal milling plant fractions such as Dorrcone centrifugestreams, Clari-fier under-flow and de-germed primary mill streamslurries. Various other crude starchy materials with which the art isfamiliar may likewise be used.

It is essential in the practice of this invention that the starchgranules are in such form that the amyloglucosidase can contact andattack the starch molecule to convert it to the dextrose vmolecule. Insome instances this rupturing of the saroh granule may be brought aboutby prior manufacturing operations. Then the amyloglucosidase enzyme maybe mixed directly with the amylaceous material. It is preferred however,to insure the availability of the starch molecule to action by theamyloglucosidase by a pre-treatment step which involves contacting theamylaceous material with a liquefying enzyme such as a protease-freeamylase under conditions that will insure rupture of the starch granule.I

This operation. referred to as liquefaction, is accomplished bycontacting the amylaceous material with an amylase enzyme, for example,at a temperature within the range of from about 140 F. to about 195 F.,for from about 20 to 40 minutes. Although this liquefaction step may becarried out in an agitator vessel equipped with means for temperaturecontrol, it also may be ac complished in a flash sterilizer, a Votator,or in other equipment which is known to the art. It has been found thatuse of higher temperatures decreases the holding time substantially.Care should be taken, however, not to raise the temperature of theliquefaction step to one which causes inactivation of the liquefyingenzyme. The liquefaction step is preferably carried out at a pH ofwithin the range of 6.5 to 7.5.

The amount of liquefying enzyme utilized will depend upon the potency ofthe material used. Ordinarily from about 0.06% to about 0.15% by weightbased on the weight of the dry solids in the amylaceous material issatisfactory when the liquefying enzyme has a potency expressed inmilligram equivalents of starch conversion to moltose under standardconditions of from about 40 to 100.

If it is found desirable to use the liquefaction step, such liquefyingenzymes are preferably inactivated prior to the subsequent treatmentwith amyloglucosidase. This may be done by heating to a temperaturesufficient to inactivate the liquefying enzyme, for example, to about210. F.

Conversion of the liquefied amylaceous material to dextrose is broughtabout by contact with the amyloglucosidase enzyme system underconditions which are conducive to this conversion. This conversion iscommonly referred to as the saccharification step. The temperature ofthe liquefied material is adjusted to one within the range of from about130 F. to 145 F., and the pH adjusted to between about pH 4.0 to pH 5.5.There is then added to the liquefied mixture sufficient amyloglucosidaseto convert the starch present to dextrose. The amount of enzyme addedwill again depend upon enzyme potency. Nonmally from about 0.05% to0.10% by weight, based on the weight of the dry solids in the originalmixture, is sufiicient. Contact between the liquefied starch and theamyloglucosidase enzyme system is maintained for about 45 hours to hoursand the temperature controlled to one within the range of from about F.to F.

After the conversion of the starch to dextrose is substantiallycompleted the mixture may then be filtered to remove solids and theresulting filtrate containing the dextrose solution may be furtherpurified and concentrated to a water content of between 25 and 16percent prior to crystallization to recover the dextrose in solid formor the dextrose solution may be used directly in any of the numerousindustrial applications with which the art is familiar.

The following examples are included for the purpose of illustrating myinvention.

Example 1.-Refined dry amyloglucosidase p0wder use of fullers earth Toone liter of a filtered, submerged culture of Aspergil- Zus nigercontaining 2300 amylogluoosidase units was added 50 grams of fullersearth. The mixture was stirred and the pH of the mixture was adjusted topH 4.0 with hydrochloric acid. After stirring for 30 minutes, themixture was filtered and evaporated under. reduced pressure at 40 C. toa volume of 200 cc. 400 cc. of isopropanol was added and the mixturestirred vigorously and allowed to stand until a gummy precipitateformed. This precipitate weighing 30 grams, which contained the purifiedamyloglucosidase, settled to the bottom of the container.

The 30 grams of isolated amyloglucosidase concentrate was blended with150 grams of dry, powdered cornstarch and mixed until a damp, friableproduct resulted. This product was then air-dried and ground to producea light colored, free-flowing powder that weighed 180 grams.Accordingly, 0.18 gram of the powdered amyloglucosidase productrepresented 1.0 cc. of the original culture.

Example 2.- Refined liquid amylglucosidase use of fullers earth Thirtygrams of gummy precipitate obtained as in Example 1 was dissolved in 100cc. of water. The resulting solution was filtered and placed in arefrigerator.

This amyloglucosidase preparation contained about 90 percent of theamyloglucosidase activity of the original culture liquor. It wassubstantially free from protease, lipase and transglucosidaseactivities.

Example 3.-Refined amyloglucosidase s0luti0n use 0 magnesium silicate Anamyloglucosidase preparation substantially free from protease, lipase,transglucosidase, and other carbohydrases such as alpha-amylase andbeta-amylase was prepared as follows: 20 grams of magnesium silicatesoid under the trade name of Mag'nesol was added to one liter of afiltered, submerged culture liquor of Aspergillus niger at pH 4.0. Themixture was stirred at room temperature for 30 minutes, and filtered.The clear filtrate was exaporated at pit-I 4.0 under vacuum at 35 C. to40 C. to a volume of 500 cc. An equal volume of acetone was added withvigorous stirring at room temperature.

The mixture was allowed to stand until a precipitate formed. Theprecipitate was dissolved in 250 cc. of water and filtered. To thisliquor was added an equal volume of acetone and the stirred mixturecentrifuged. The sediment was discarded. 1.0 gram of sodium chloride wasadded to the clarified liquor and the mixture stirred until the salt wasdissolved. The solution was allowed to stand until a fiocculantprecipitate formed.

The mixture was centrifuged and the clear liquor dis.- carded, Theprecipitate was mixed with 100 cc. of water and centrifuged. The clearwater-white solution contained the purified amyloglucosidase. Thisrefined amyloglucosidase solution was placed in a refrigerator untilused.

Example 4.-Refined amyloglucosidase s0luti0n-use of bentonite clay 4.0grams of bentonite clay was added to one liter of filtered,submerged-culture liquor of Aspergillus niger. The mixture was stirredand the pH maintained within the range of about pH 3.8 to pH 4.3 byadding hydrochloric acid. After 30. minutes of stirring the mixture wascenterifuged. The clear centrifuged liquor contained from 90 to 95percent of the original .amyloglucosidase activity, whereas about 90percent of the transglucosidase activity was removed by the bentoniteclay.

Example 5 .Preparation of isolated amyloglucosidase One liter of a crudefungal extract produced by means of an Aspergillus niger culture inaccordance with procedures known to the art containing 2300amyloglucosidase units was subjected to a refining and isolationtreatment according to Example 1.

Amyloglucosidase activity units were determined on the dry powderedproduct according to the-procedure hereinabove given, using 0.20 gram ofthe powder per 5 grams of soluble starch. The amyloglyucosidase activitywas found to be 13.8 grams per gram of the powdered prodnot. That is tosay, one gram of the powdered enzyme product was calculated to contain13.8 amyloglucosidase uni-ts, which is equivalent to saying that onegram of the powdered product could produce in this test 13.8 grams ofreducing sugar calculated as dextrose.

' method of Sicher-t and Bleyer.

The absence of harmful amounts of transglucosida-se was shown in thefollowing test: A sample of 50.0 grams, dry basis, of Lintners solublestarch was dissolved in sufiicient water, by heating, to give a 35.0%solution at pH 4.0, to which was added 0.45 gram of the powdered enzyrnepreparation at 60 C. Hydrolysis was continued at 60 C. for 72' hours.The hydrolyzate at this time was found to have a pH of 4:1. It.w-asfiltered, heated in a boiling water bath for 15 minutes and cooled. TheD.E. was determined by the Schoorl test on an aliquot of the hydrolyzateand true dextrose was determined by the The D.E. was found to be 94% andtrue dextrose, 92% dry basis. Obviously, as will be demonstrated byspecific test subsequently, little if any transglucosidase was presentin the processed enzyme preparation since the DE. found was only 2% morethan the percentage of true dextrose present; that is, very nearly allof the reducing sugars in the hydrolyzate was actually dextrose.

Quite -in contrast, when an identical hydrolysis procedure was carriedout using an equivalent amount of the original crude extract tohydrolyze the starch, then after 72 hours at 60 C., the hydrolyzateanalyzed DE. and only 82.0% true dextrose, dry basis. Therefore, 8% lesstrue dextrose actually resulted when the crude extract was used, andfrom the much wider spread between DE. and true dextrose values thanwhen the processed enzyme was used, it is apparent that a very largepart of this 8% loss in dextrose yield was due to production ofsynthetic sugars from dextrose by transgluco-sidase in the crudeextract.

This last test also shows the apparent gain in amyloglucosidase activitymerely by processing the enzyme preparation.

This last test shows furthermore that 0.45 gram of dry enzyme powder wasable to produce 45 grams of true dextrose (from 50 grams of starch).Inasmuch as 0.45 gram of the enzyme powder contained 6.21amyloglucosidase units, then one unit of this powder was able to producevery nearly 9 grams of dextrose, when given sufficient time and a highersubstrate concentration (35%) than is used in the amylglucosidase unitstest, described hereinabove. I Actually, therefore, the dry enzymepowder (which is mostly inert carrier of starch in granule form) canhydrolyze about times its weight of dissolved starch to dextrose.

Transglucosidase activity was determined on the dry powder enzymeproduct by the procedures hereinabove given. The value found was only0.03 gram of nonfermentable substance per amylglucosidase unit; incontrast, the crude original extract had a transglucosidase activity of0.18 gram of non-fermenta'ble per amylglucosidase unit. This six-folddifference in transglucosidase activity readily explains the 8% lesserdextrose yield in using the crude extract to hydrolyze starch. For ifonly a little more than half of the non-dextrose reducing sugars weresynthetic non-fermentables when the isolated amyloglucosidase was used(or about 1.2%), then six times this value, or 7.2% of synthetics, plusabout 0.8% of natural non-dextrose reducing sugars, would account for 8%lesser dextrose yield.

Substantially no protease or lipase activity was found in the processed,dry enzyme powder, when it was tested 'by the usual procedures known tothe prior art.

My invention enables for the first time, the introduction of in situdextrose into a starch-containing food product, without modifying orchanging in any way the nonstarch ingredients of the said food. In somecases, it may be desirable to convert only a minor portion of the starchin the food to dextrose. In other instances, certain advantages arecreated by subjecting the starchy food to amyloglucosidase convertingconditions wherein all, or a major portion of the starch in the food, isconverted to dextrose. The particular degree of dextrose conversion willdepend on the ultimate end-use which is intended for the saidenzyme-converted food.

In the case of tamylaceous products which contain enzymes emanating fromeither the growing, harvesting, or steeping of these materials, the saidproducts, before amyloglucosidase conversion, can either be subjected toa sufficiently high temperature to destroy these natural enzymes, ortreated directly with amyloglucosidase without any predestruction of thenatural enzymes. The particular sequence of any amyloglucosidas'etreatment will depend upon the nature of the ultimate end-product afterthe amy-loglucosidase treatment. Thus, in the case of bread dough, it isinadvisable to preheat the wheat flour to a temperature that woulddestroy the natural enzymes of the Wheat. In the case of corn or ricefor use as raw material for finished slab dextrose sugar, it isimportant to destroy all the natural enzymes in these cereals beforeproceeding with the amyloglucosidase treatment.

Examples of edible amylaceous products which can be advantageouslysubjected to my enzymatic in situ producing process as described aboveare various products of the dry cereal milling industry, wet cornmil-ling products, vegetables and starchy fruits. Some specific examplesof the above generic types of food products which could beadvantageously subjected to the processes of this invention are wheatflours, rice, tapioca, whole ground cereals such as corn or barley;cornmeals, hominy feed, and various wet corn milling industry fractions,such as mill starch, corn gluten, gluten meal, squeezer slop and variousmillstream fnactions such as Merco centrifugal liquors, or starchliquors going to or coming from Dorrcone equipment. Crude corn sugarliquors, corn syrups or malt syrups may also be advantageously subjectedto a treatment with amyloglucosidase which is free of transglucosidase,protease and lipase.

For the attainment of food products of enhanced dextrose content,fruits, or vegetables, after proper com niinuation or fragmentation, maybe blended into a water slurry of finely ground cereals, degerminatedcereals, refined starch, or mixtures thereof. A subsequent refinedamyloglucosidase conversion, followed by suitable dehydration, willresult in a dry food comprising in situ dextrose intimately blended withthe non-dextrose solids of the said vegetables or fruits.

Example 6 A wet cornmilling crude starch liquor fraction coming from theMerco centrifugal of the millhouse and containing 2.0% of protein, basedupon the dry starch content of said liquor, was subjected to acentrifugal washing treatment in a Dorrcone centrifugal of the type nowin common use in the wet corn milling industry. The washing was donewith warm, 130 F. fresh water, and was sufficient to remove the majorportion of water-soluble impurities which contribute towards deleterioustaste and flavor effects when such a millhouse starch product isintended for food end-use purposes.

The washed Dorrcone starch liquor was adjusted to a starch content of35% and then blended with 0.1%, based upon dry starch content, of aprotease-free liquefying amylase, i.e., a liquefying amylase which isfree of protease activity at starch pasting and liquefactiontemperatures, such as the product produced by Miles Laboratories, Inc.,under the trade name of HT 440.

The starch liquor was then heated to a suflicient temperature to pastethe corn starch and cause a substantial liquefaction of the said paste,with a minimum of saccharification. The resulting liquefied starch pastewas then heated to a boil to inactivate the liquefying amylase, cooledto 140 F., and the pH adjusted to 4.1. A blend of amyloglucosidase syrupand starch, produced as described in Example 1, was then added to thispaste in an amount corresponding to 1.0%, based upon the dry starchcontent of the paste.

The amyloglucosidase was allowed to act upon the above paste under thesaid temperature and pH conditions for a period of hours. This resultedin the conversion of 96% of the starch to dextrose. This liquor wasadjusted with soda ash to pH 5.0 and centrifuged. The resulting dextroseliquor Was vacuum concentrated to a water content of 17%. Theconcentrated liquor was then poured into a seeding tank containing 3% ofdextrose hydrate crystals. The batch was thoroughly blended, cooled intwo hours to 100 F., and poured onto a concrete floor to a depth of sixinches. The concentrated mass of dextrose liquor was allowed to cool andcure at room temperature. This resulted in the production of a solidslab of high quality dextrose hydrate sugar which was free of bittertasting dextrose impurities.

In contrast to the old slab corn sugars known to the prior art andproduced from refined corn starch by means of acid hydrolysis, my newtype of slab corn sugar, when tested in accordance with the analyticalprocedures submitted in this specification, was found to besubstantially free of gentiobiose, panose, other poor tasting ornonfermenting sugars, or protein breakdown products caused by enzymaticdegradation.

When ground to a fine mesh, the above slab sugar was found to be apleasant tasting material which could be substituted for isolatedcrystalline sugars such as dextrose or sucrose in many end-uses whereinthe slab or chip corn sugar known to the prior art was incapable ofacceptance as a crystalline sugar substitute.

The absence of carbohydrate polymers in the concentrated dextroseliquors produced by my process, minimizes dextrose crystallzationretardation caused by inhibiting colloids, and enables a much more rapidcrystallization and concomitant slab formation of my improved slab cornsugar product.

It is preferable to use a small amount of protease-free liquefyingamylase before applying the amyloglucosidase to the starch paste. Theliquefying step enables the use of starch pastes of greater drysubstance content, and thereby lowers vacuum pan dehydrating costs.However, one may, if so desired, paste the starch without any anzymaticliquefaction treatment, andsubject said paste upon cooling to about F.,to the amyloglucosidase treatment.

The amount of dextrose added for crystal seeding per pound will vary,depending on the amount of non-dextrose ingredients, the non-dextrosesolubles, and the rate of cooling of the blend with the concentrateddextrose liquor. With pure starch as the amyloglucosidase substrate,little or no dextrose seed would be required, depending upon the timeallowed for slab sugar formation.

In general the protease-free amylase treatment may be accomplished at atemperature Within the range of about 140 F. to 195 F. in about 20 to 40minutes and at a pH of preferably from about 6.5 to 7.5. Followingliquefaction, the liquefied starch solution is generally cooled to atemperature within the range of about 130 to F. and the pH adjusted toabout from pH 4.0 to pH 5.5. The amyloglucosidase conversion may beeffectively conducted at temperatures in the range of about from 130 F.to 145 F. and in about 45 to 90 hours.

Example 7 A Wheat flour containing 12 percent protein was subjected to adry milling sifting and aspirator procedure under conditions whichresulted in the production of a wheat flour containing 2.2 percentprotein.

The low protein content Wheat flour was suspended with sufiicient coldwater to produce a 35 percent starch milk. The resulting starch liquorWas then blended with 0.1 percent, based upon dry flour content, of theHT-440 liquefying enzyme, and subjected to rapid liquefying treatment ina Votator under conditions wherein the starch liquor was heated to F. in30 minutes. The paste was then held at 190 F. for 10 minutes, heatedrapidly to 210 F., cooled to 140 F., .and then subjected to the sameamyloglucosidase and crystallizing conditions as that described inExample 6.

Upon grinding the resulting slab wheat dextrose to a fine powder, therewas obtained a pleasant tasting sugar product which also had within itthe flavoring efiects of the non-modified, non-starch constituents ofthe low protein wheat flour.

This type of wheat sugar product is suitable for end- .uses involvingthe use of isolated crystalline sugars such as sucrose or dextrose, inconjunction with various types of wheat flours or doughs.

Because of the low protein content of the above wheat flour, it ispossible to readily paste the flour and to process the resulting pastewith amyloglucosidase, without inviting diificulties from lumping ordoughing effects caused by the presence of normal amounts of wheatgluten in wheat flour.

With proper facilities for overcoming the pasting and dehydratingdifficulties caused by the presence of wheat gluten, a regularwheatflour with normal protein can also be processed into a slab wheat sugarproduct.

Example 8 Ground whole corn was mixed with sufiicient water to produce aslurry containing 33 percent of dry substance. The starch was rapidlyliquefied with protease-free alpha amylase in a continuous paster, suchas a Votator, and heated to 210 F. The hot liquefied paste was cooled to140 F. and was adjusted to pH 4.0. The paste was converted withamyloglucosidase under the same conditions as described in Example 6.

During the conversion, some of the corn oil in the, corn was liberatedand floated on top of the slurry. After the dextrose conversion, theliquor was adjusted to pH 5.0 with soda ash.

The liberated corn oil was skimmed off and the crude dextrose liquorcontaining suspended protein, germ and fibre material was centrifuged.Centrifuged liquor was then dehydrated and subjected to slab sugarformation as described in Example 6. The residue obtained from thecentrifuging consisted of a material of high protein content. Thismaterial was valuable for upgrading the protein content in corn glutenfeed or in other animal feed products.

The slab corn sugar obtained from the centrifuged liquor had taste andfermentability qualities similar to that obtained from the Merco starchliquor described in Example 6.

' Since ground corn is a cheaper starting material, the slab sugarobtained in accordance with this example is a lower cost product whichcan be used to advantage by the brewing industry in place of crude corngrits In this example the solubles present in the original whole cornend up in the slab sugar. These solubles contribute toward certainflavor effects which, with some food end-uses, are favorable. However,where the prime objective is maximum in situ dextrose content with aminimum of non-dextrose solubles, it is preferable to start with anamylaceous raw material in the solubles of which have been removedbefore the amyloglucosidase treatment.

Example 9 Hominy feed was suspended in warm, 130 F. water, stirred for30 minutes, and centrifuged. The washed centrifuged cake, free of itscorn solubles, was resuspended in fresh water and subjected to thetreatment described in Example 8.

There resulted a slab corn sugar, the taste of which was not affected bythe water soluble ingredients present in the original corn kernel.

Example 10 In a dry corn milling plant, the stream or channel of hominygrits intended for hominy feed production was removed before theincorporation of corn germ and corn breakdown products.

bran fractions which are commonly added to the hominy grits to producehominy feed.

The said hominy grits were then subjected to the same treatment as thatdescribed in Example 8, with the exception of the omission of thecentrifuging of the dextrose liquor resulting from the amyloglucosidascconversion of the starch into grits.

There resulted a low cost, converted corn grits product which isparticularly suitable as a replacement for either crude corn grits orrefined corn starch grits which are now being used by the brewingindustry. The advantage of my new type of modified brewers grits arelower cost coupled with maximum solubles extract and maximum yeastfermentation efficiency.

The product produced per this example has similar advantages when usedinother fermentation industries, such as vinegar, alcohol, or lacticacid.

In the case of intended end-uses wherein the Water solubles present inthe untreated hominy grits are undesirable, the hominy grits can besubjected to a preliminary warm water wash and centrifuging step toremove the said solubles. For such procedure, the preferred arrangementis to have the hominy grits processed in the millhouse of a wet cornmilling plant. This enables the extracted solubles from the hominy gritsto be processed into corn gluten feed, steep water, or gluten mealchannels, thereby obviating the need for'taking an economic loss on thesolubles removed from the hominy grlts. In place of a wet corn millingplant, similar advantages would accrue by having the hominy gritsprocessed in a yeast manufacturing plant, alcohol or whiskeydistilleries, or sugar manufacturers, such as cane or. beet sugarrefineries.

Example 11 A wet corn milling liquor coming from the Dorrconecentrifugal of the millhouse, and intended as the starting starchmaterial for corn syrup production, was subjected to the same treatmentas that described in Example 6,.with the exception that the concentrateddextrose liquor in the vacuum pan was not crystallized into slab sugar,but instead of this, was blended with a sufficient amount of commercial,refined, invert sugar to prevent the dextrose from crystallizing in adegree sufficient to cause the formation of a solid slab sugar.

The resulting product was a thick syrup comprising dextrose andlevulose, and was free of maltose, d'extrins, retrog-radation dextrosepolymers and enzymatic protein My improved syrup form dextrose productmakes possible the use of dextrose in various food products without theneed for first isolating the dextrose in crystalline form, drying thesaid crystals and then redissolving the said dried crystals to produce adextrose solution.

Example 12 A refined corn syrup 'was mixed with sufficient water toincrease the water content of the syrup to 40 percent. The water usedfor the addition to the corn syrup was first blended with a sufficientamount of amyloglucosidase product of the kind described under Example1, to represent 1.0 percent of the non-dextrose carbohydrate content ofthe corn syrup. The diluted corn syrup was adjusted to pH 4.0 andallowed to stand at room temperature. After standing for 30 days, thenon-dextrose carbohydrates in the corn syrup were converted to dextrose,and a solid mass of dextrose sugar resulted.

This solid dextrose sugar contained whatever gentiosw m 13 corn starch.The fatty acids appear in a form of what is known in the wet cornmilling trade as refinery mud.

To obtain a dextrose syrup similar to that described in Example 11, butfree of refinery mud and substantially free of coloring matter, thediluted corn syrup of the present example may be blended With refinedliquid invert sugar just prior to the time when the amyloglucosidasecreates a suificient amount of dextrose .to cause solidification.

The exploitation of my corn syrup amyloglucosidase treating procedurecan be advantageously used by candy manufacturing plants, by storingcorn syrup in tanks, subjecting this syrup to the said amyloglucosidasetreatment, and then producing the required batch of dextrose liquor byleaching the solidified solid dextrose sugar resulting from theenzymatic treatment of the corn syrup, withhot water, and adjusting thepH of the sugar solution to pH 4.8.

To accelerate dextrose crystallization in the amyloglucosidase treatedcorn syrup, the batch maybeblended with some ground slab dextroseproduced from previous batches or with pure, crystalline dextrosehydrate or anhydrous dextrose.

The dextrose product obtained per this example still contains thedextrose polymers created by the reversion action of dextrose during theacid hydrolysis treatment of the corn syrup. This product, therefore,cannot approach as closely the taste qualities of isolated crystallinedextrose as is possible by using pure corn starch as theamyloglucosidase substrate. However, this solidified dextrose obtainedfrom corn syrup can be used in many confectionery, ice cream, canning,and other food uses wherein the small amount of dextrose impuritiespresent in this amyloglucosidase converted corn syrup are insufiicientto seriously interfere with the ultimate taste and flavor of thefinished food.

If it is desired to obtain a corn syrup more amenable to subsequentdextrose formation without parallel transglucosidase formation adiastatically prepared syrup may be used. Thus, a water-washeddegerminated corn meal is subjected to an in situ amylase conversion toproduce a refined syrup consisting of maltose, dextrins and somedextrose. This can be used by large dextrose consumers such as bakers orconfectioners to convert the said syrup entirely to dextrose bycontinuing the refined amyloglucosidase conversion.

The unique ability of isolated amyloglucosidase to function inconcentrated carbohydrate solutions is what makes possible the treatmentof corn syrup at high carbohydrate concentration. This, together withthe ability of amyloglucosidase to function tangibly at lowtemperatures, such as 60 F makes it possible to develop a processwherein convertabledextrose polymers are changed to dextrose byamyloglucosidase under cost-saving processing conditions.

Example 13 A refined corn starch which had been thoroughly washed withwarm, 130 F. fresh water to remove sub stantially all of the watersoluble impurities, was resuspended in fresh water at a starchconcentration of 35 percent. To this slurry of highly refined starch,there was added 0.1 percent, based upon dry star-ch content, of theHT-440 liquefying enzyme, described in Example 6. The blended slurry wasthen subjected to a rapid paste liquefying treatment in a continuouspastor of the Votator type under conditions wherein the starch liquorwas heated to 190 F. in 30 minutes. The paste was then held at 190 F.for minutes, heated rapidly to boiling to inactivate the enzymematerial, and cooled to 140 F.

The cooled paste was adjusted to pH 4.0 and 1.0 percent of theamylogluoosidase enzyme preparation, described in Example 1 was added.The paste was then converted for 90 hours at 140 F. This produced aliquor analyzing 97.1 percent DE. and 95.5 percent actual dextrosecontent.

The pH of the resulting dextrose liquor was adjusted with soda ash to pH4.9, and an amount of Darcodecol orizing carbon corresponding to 1.5percent of the dextrose content was added to the liquor. The liquor washeated to 150 F. and after a 30 minute stirring of the decolorizingcarbon suspension was filtered and the filtrate vacuum concentrated to awater content of 16 percent. The concentrated liquor was seeded with 1.0percent of dextrose hydrate and thoroughly blended. The seeded liquorWas cooled to room temperature and then poured into cotton bags. Uponstanding at room temperature for a period of 12 hours, the dextrosesugar in the cotton bags solidified into a solid cylindrical mass.

These cylindrical masses of bagged solid dextrose were then used at asugar-consuming plant, such as a candy factory, by rolling thecylindrical bags of dextrose into tanks containing fresh hot water, andallowing the hot water to dissolve out the dextrose from the cottonbags. The leached-out cotton bags were then recovered from the sugarsolution tank, dried, and were then ready for re-use for the formationof other cylinders of solid dextrose.

The product obtained in accordance with this example is one the qualityof which approaches that of pure crystalline dextrose hydrate from thestandpoint of taste as well as fermentability.

By practicing the process of this example, my invention makes possiblethe production of a substantially pure dextrose hydrate product withoutresorting to the much more expensive procedures that are necessary forthe production of isolated crystalline dextrose from acid-convertedstarch liquors. My process eliminates acid conversion in pressurecookers, long periods of dextrose crystallizing in expensive sugarcryst-allizer equipment, sugar centrifuging of the crystals, and sugardriers to dry the crystals.

The absence of transglucosidase in my enzyme converting procedure makespossible the production of a solid, slab dextrose product, the actualdextrose content of which is substantially greater than heretoforepossible. Since the higher the dextrose content, the lower the amount ofnon-dextrose impurities, the ability to produce, with my process, aproduct containing over percent of actual dextrose, enables the creationof an insitu dextrose hydrate having taste and color qualitiesapproaching that of isolated crystalline dextrose.

The absence of any substantial amounts of non-dextrose polymer materialmakes possible, with my process, a radical reduction in the time neededfor solidification to rigid -slab form. The crude slab dextrose productsof the past required several days of curing before a rigid slab wasobtained. In contrast, my process enables slab formation in a few hours.To reduce the slab formation time still farther, the cotton bag used toform the cylinders of slab dextrose, should be equipped with a series ornest of aluminum water-cooling coils. Upon circulating a cooling liquidthrough the said cooling coils, the time for producing a solidcylindrical slab of dextrose in the cotton bag may be reduced to aslittle as two hours.

In place of cotton bags, any flexible polymer fibre container capable ofeasy hot water penetration, and being inert toward hot sugar solutions,can be used for the production of cylindrical slabs of sugar from otheramylaceous materials, such as degerminated corn and other cerealproducts.

In a brewery, the use of a portabrle, rollable, cylindrical slab ofdextrose made from degerminated corn in accordance with my procedure,enables a greatly simplified handling of fermentable carbohydrates bythe brewer and eliminates the moving of large volumes of dusty corngrits by means of expensive grain handling equipment.

Example 14 grams of wheat flour were suspended in 500 cc. of 77 F.water. The resulting aqueous wheat flour susperiod of 3 and 5 hours,respectively. The dextrose content of the enzyme-modified wheat flourwas deter-:

mined at the end ofeach of the above said converting times.

It was found that, after 3 hours, the enzyme-treated wheat flour had 3.4percent dextrose, and that after 5 hours the wheat flour contained 4.3percent dextrose, both dextrose figures being based upon a moisture freewheat flour.

Example Five parts by' weight of wheat flour were suspended in 100 partsby weight of water and the mixture pasted by heating to 190 F. The hotpaste was then cooled to 77 F.- and there was added 0.05 part by weightof amyloglucosidase enzyme having a potency of 13.8 amylo- 'glucosidaseunits per gram. The mixture was allowed to convert for 6 hours at 77 F.After this converting time the pasted wheat flour, on an originalmoisture-free dry flour-basis, contained 5.4 percent dextrose.

65 parts by weight of the above dextrose-containing dilute pasted wheatflour was then used as the source for water and sugar in a white breadformula based upon the following proportions of ingredients:

Ingredient: Percent on flour basis Flour 100 Pasted wheat flour liquorcontaining dextrose 65 Amyloglucosidase enzyme 0.5 Yeast 2 Yeast foo-d0.4 Non-fat milk solids 5 Shortening 4 The above white bread formula was.then used in a regular sponge dough method baking procedure wherein thefermentation time was four hours and fermentation temperature was 77 F.

It was found that under the above conditions the total amount ofdextrose sugar created in situ was 5.8 percent based upon weight of thecommercial flour. This is about the same amount of sugar, particularlythe commercial crystalline, refined dextrose hydrate sugar,whichiscommonly added by commercial bakers for the purpose of acting asa yeast food during the fermentation time.

In Examples 14 and 15, in situ dextrose production was created in thewheat flour under temperature, pH, and fermenting time conditions whichcorrespond to the conditions prevailing in commercial bread baking. Thisenables dextrose production for the yeast fermentation withoutinterfering with the regular bread baking procedure.

In Example 14, where the amylogucosidase was added to ungelatinizedwheat flour, the ability of the enzyme to create dextrose fromungelatinized material is unique. The reasons for this unusualamyloglucosidase action are not completely understood, but it ispresumed that commercial wheat flour, during the dry milling process,has some of the wheat starch granules subjected to sufficient mechanicaldisintegration to enable in situ production of dextrose by the enzyme.

Instead of bread, my in situ dextrose procedure may be similarly appliedto other bakery products, such as cakes, sweet rolls, doughnuts, and thelike.

It will be appreciated by those skilled in the art that manymodifications can be made without departing from the principlesformulated by this invention. The

dextrose-producing enzyme need not be limited to amyloglucosidaseobtained from fungal sources. Any amylase which is free of protease,lipase, and transglucosidase, and which has the property of producingexclusively dextrose from starch is applicable to this invention. Forthe paste-converting conditions for dextrose lization retardation.

The slabs or cylinders of solidified dextrose may have incorporatedwithin them, before solidification, other food products, such asflavoring materials, milk solids, egg solids, or the like. Such blendsaresuitable for certain candy, bakery, and similar food productoperations.

The procedure described for obtaining cylindrical bags of solid dextrosefrom a solidifiable massecuite, is applicable to other sugars such assucrose, lactose, or maltose, and the like.

In view of the foregoing disclosure, those skilled in the art will beable to practice the invention either by following the embodiments givenor such other embodimentsor modifications of the invention as will beapparent. Accordingly, all matter disclosed above is intended to beinterpreted as illustrative and not in a limiting sense.

What is claimed is: 1. A process for the prepartion of dextrose from acrude starch containing amylaceous material containing protein and fat,which comprises the steps of subjecting protein and fat which comprisesthe steps of subjecting said crude starch material to the action of aprotease-free amylase at a temperature within the range of from about F.to 195 F. for. from 20 to 40 minutes and at a pH of from 6.5 to 7.5 toliquefy substantially all the starch in said crude material, cooling theliquefied mixture to a temperature within the range of from about 130 F.to F., adjusting the pH of said liquefied mixture to one within therange of pH 4.0 to pH 5.5, adding thereto an amyloglucosidase which issubstantially free from protease, lipase and transglucosidase activity,and maintaining said mixture at a temperature within a range of fromabout 130 F. to 145 F. for from about 45 to 90 hours with mild agitationto convert said starch to dextrose.

3.A process for the preparation of dextrose from a crude starchcontaining amylaceous material containing protein and fat, whichcomprises the steps of subjecting said crude material to the action of aprotease-free liquefying amylase under liquefying conditions to liquefysubstantially all of the starch in said crude material, subjecting theliquefied mixture to the action of an amyloglucosidase which issubstantially free from protease, lipase and transglucosidase activityso as to convert the liquefied starch to liquid dextrose solution,separating said dextrose solution from the insoluble portions of saidmlxture, concentrating said dextrose solution, and crystallizingdextrose from said dextrose solution.

4. A process for the prepartion of dextrose from a crude starchcontaining amylaceous material containing protein and fat, whichcomprises the steps of subjecting said material to a water washingtreatment to remove water soluble constituents thereof, subjecting thewashed crude material to the action of a protease-free liquefyingamylase under liquefying conditions to liquefy substantially all of thestarch in said crude material, subjecting the liquefied mixture to theaction of an amyloglucosidase which is substantially tree from protease,lipase and transglucosidase activity so as to convert the liquefiedstarch to liquid dextrose solution, separating said dextrose solutionfrom the insoluble portions of said mixture, concentrating said dextrosesolution, and crystallizing dextrose from said dextrose solution.

5. A process for the preparation of dextrose which comprises subjectinga Wet cornmilling crude starch fraction to a water Washing treatment toremove water soluble constituents thereof, subjecting said washed crudestarch fraction to the action of a protease-free liquefying amylaseunder liquefying conditions to liquefy substantially all of the starchin said crude starch fraction, subjecting the liquefied mixture to theaction of an amyloglucosidase which is substantially free from protease,lipase and transglucosidase activity so as to convert the liquefiedstarch to liquid dextrose solution, separating said dextrose solutionfrom the insoluble portions of said mixture, concentrating said dextrosesolution, and crystallizing dextrose from said dextrose solution.

6. A method for the enzymatic production of a solidified crystallinedextrose composition from a crude starch containing amylaceous foodmaterial containing at least one non-starch constitutent selected fromthe class consisting of proteins and fats Without the simultaneousproduction of conversion products caused by enzymatic action on theabove non-starch constituents present in said food and without thesimultaneous production of non-fermentable dextrose polymers, comprisingforming a pasted aqueous slurry of said food in the presence of aprotease-free, lipase-free liquefying amylase, heating the liquefiedpaste to inactivate enzyme materials, cooling the paste to anamyloglucosidase converting temperature, converting over 90 weightpercent of the starch content of the crude amylaceous food material todextrose by means of an amyloglucosidase substantially free of protease,lipase and \transglucosi-dase, dehydrating the resulting dextrosesolution free of protein and fat enzyme conversion products and free ofnon-fermentable dextrose polymers to a water content of between 25 and16 percent, cooling the resulting concentrated dextrose solution, andallowing said cooled solution to stand until solidification takes place.

7. A method for producing dextrose from at least a portion of the starchcontained within the starch granules of a crude amylaceous material,said crude amylaceous material also containing at least one non-starchconstituent selected from the class consisting of proteins and fats,which comprises rupturing at least a portion of the starch granules ofthe crude amylaceous material to an amyloglucosidase-convertible stateand subjecting said ruptured starch granules to the action of anamyloglucosidase which is substantially free from protease, lipase andtransglucosidase activity thereby converting at least a portion of thestarch to a dextrose-containing product which is substantially free ofprotease-converted products, lipaseconverted products,transglucosidase-converted dextrose polymers and acid-hydrolyzedproducts.

References Cited by the Examiner UNITED STATES PATENTS 1,773,296 8/1920Block 99-80 2,365,788 12/1944 Warburton 99-142 2,822,303 2/1958 Campbellet al. 99-142 X 2,842,442 7/ 1958 Jefireys 99-90 2,881,115 4/1959Liggitt et al. -66 2,891,869 6/1959 Langlois 99-142 2,893,921 7/1959Langlois et al 195-66 2,967,804 1/1961 Kerr 195-66 3,017,330 1/1962 Kerr195-66 X 3,039,936 6/1962 Lenney et al. 195-11 A. LOUIS MONACELL,Primary Examiner.

R. N. JONES, Assistant Examiner.

7. A METHOD FOR PRODUCING DEXTROSE FROM AT LEAST A PORTION OF THE STARCHCONTAINED WITHIN THE STARCH GRANULES OF A CRUDE AMYLACEOUS MATERIAL,SAID CRUDE AMYLACEOUS MATERIAL ALSO CONTAINING AT LEAST ONE NON-STARCHCONSTITUENT SELECTED FROM THE CLASS CONSISTING OF PROTEINS AND FATS,WHICH COMPRISES RUPTURING AT LEAST A PORTION OF THE STARCH GRANULES OFTHE CRUDE AMYLACEOUS MATERIAL TO AN AMYLOGLUCOSIDASE-CONVERTIBLE STATEAND SUBJECTING SAID RUPTURED STARCH GRANULES TO THE ACTION OF ANAMYLOGLUCOSIDASE WHICH IS SUBSTANTIALLY FREE FROM PROTEASE, LIPASE ANDTRANSGLUCOSIDASE ACTIVITY THEREBY CONVERTING AT LEAST A PORTION OF THESTARCH TO A DEXTROSE-CONTAINING PRODUCT WHICH IS SUBSTANTIALLY FREE OFPROTEASE-CONVERTED PRODUCTS, LIPASECONVERTED PRODUCTS,TRANSGLUCOSIDASE-CONVERTED DEXTROSE POLYMERS AND ACID-HYDROLYZEDPRODUCTS.